Metabolic activation system

Results of methyl parathion assays involving effects on chromosomes have also been contradictory. For sister chromatid exchange, Waters et al. (1982) reported a positive response in Chinese hamster ovary cells only in the presence of metabolic activation system, while methyl parathion tested positive without a metabolic activation system in Chinese hamster V79 cells (Chen et al. 1981), cultured normal human lymphoid cells (Chen et al. 1981 Gomez-Arroyo et al. 1987 Sobti et al. 1982), and Burkitt s l5miphoma cells (Chen et al. 1981). Chen et al. (1981) found a significant dose-related increase in sister chromatid exchange in both hamster and human cultured cells, but dose-related cell cycle delays were less pronounced in human cell lines than in V79 cells. Negative results were obtained for chromosomal aberrations in human lymphocytes without a metabolic activation system (Kumar et al. 1993). 

Chen HH, Sirianni SR, Huang CC. 1982. Sister chromatid exchanges in Chinese hamster cells treated with seventeen organophosphorus compounds in the presence of a metabolic activation system. Environ Mutagen 4 621-624. 

Fort DJ, Rayburn JR, DeYoung DJ, et al. 1991. Assessing the efficacy of an Aroclor 1254-induced exogenous metabolic activation system for FETAX. Drug Chem Toxicol 14 143-160. 

In a study using cultured L5178Y mouse lymphoma cells, Rogers and Back (1981) reported that both 1,1-dimethylhydrazine and 1,2-dimethylhydrazine induced forward mutations at the thymidine kinase level in the absence of an extraneous metabolic activation system. The investigators also noted that the two dimethylhydrazines appeared to have different modes of action under these conditions. 

Fort, D.J., B.L. James, and J.A. Bantle. 1989. Evaluation of the developmental toxicity of five compounds with the frog embryo teratogenesis assay Xenopus (FETAX) and a metabolic activation system. Jour. Appl. Toxicol. 9 377-388. 

Cultured cells have a limited ability metabolically to activate some potential clastogens. This can be overcome by adding an exogenous metabolic activation system such as S9 mix to the cells (Ames et al., 1975 Natarajan et al., 1976 Maron and Ames, 1983 Madle and Obe, 1980). 

Strains/species/cell line Dose selection criteria Basis of dose selection Range hnding studies Test agent stability Metabolic activation system Controls Vehicle... 

Genotoxic Effects. No studies were located regarding the genotoxicity of 1,2-diphenylhydrazine in humans by any route of exposure. A limited number of assays have been conducted using bacteria, mammalian cell and whole animal systems. As indicated in Table 2-2, 1,2-diphenylhydrazine was mutagenic in Salmonella typhimurium, out not in Escherichia coli, and produced chromosome aberrations and sister chromatid exchanges in Chinese hamster cells. An exogenous metabolic activation system was necessary for expression of the aforementioned effects. In in vivo studies... 

Several methods for performing the test have been described, the guideline describes the diffusion and suspension test. Bacteria should be exposed to the test substance both in the presence and absence of an appropriate metabolic activation system. The response is expressed in the preferential inhibition of growth or the preferential killing of the DNA repair deficient strain. Escherichia coli polA (W3110/p3478) or Bacillus subtilis rec (H17/M45) pairs are recommended. The test should initially be performed over a broad range of concentrations. 

The simplest and most sensitive assays for detecting clastogenic (i.e. chromosomal breaking) effects involve the use of mammalian cells. Cultures of established cell lines (e.g. Chinese hamster ovary) as well as primary cell cultures (e.g. human l)nnphocyte) may be used. After exposure to a range of chemical concentrations in the presence and absence of an appropriate metabolic activation system, the cell cultures are treated with a spindle inhibitor (e.g. vinblastine) to accumulate cells in a metaphaselike stage of mitosis. Cells are harvested at appropriate times and chromosome preparations are made, stained with DNA-specific dye and the metaphase cells are analysed under the microscope for chromosome abnormalities. 

A variety of mammalian cell culture systems can be used to detect mutations induced by chemical substances. The L5178Y mouse l)nnphoma line, measuring mutation at the TK locus, is preferred. TK is an important enz)une involved in DNA synthesis. Cells are exposed to the test substance at various concentrations, in the presence and absence of a metabolic activation system, for a suitable period of time, and then subcultured to assess cytotoxicity and to allow phenotypic expression prior to mutant selection. Cells deficient in TK because of a forward mutation are resistant to the cytotoxic effects of pyrimidine analogues (antimetabolites), such as trifluorothymidine (TFT). This is because the antimetabolites cannot be incorporated into cellular nucleotides and kill the cell through inhibition of cellular metabolism. After treatment, cells are grown in a medium containing TFT mutant cells can proliferate in the presence of TFT, whereas normal cells containing TK are killed. This allows the detection of an increase in mutant... 

Busquet F et al (2008) Development of a new screening assay to identify proteratogenic substances using zebrafish danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT). Toxicol Sci 104 177-188... 

Bande JA et al (1999) Phase III interlaboratory study of FETAX, Part 3 FETAX validation using 12 compoimds with and without an exogenous metabolic activation system. J Appl Toxicol 19 447 72... 

DNA single-strand breaks were not induced by di(2-ethylhexyl) phthalate in primary cultures of rat or Syrian hamster hepatocytes or in Chinese hamster ovary (CHO) cells. Unscheduled DNA s5uithesis was not induced in rat or mouse primary hepatocytes exposed to di(2-ethylhexyl) phthalate. Only one of six studies reported induction of gene mutations at the Tk locus in mouse l5unphoma L5178Y cells exposed to di(2-ethylhexyl) phthalate in the absence of an exogenous metabolic activation system. Ouabain-resistant mutants were not induced in mouse lymphoma or BALB/c-3T3 cells. 

Chromosomal aberrations were not induced by di(2-ethylhexyl) phthalate in any of eight studies in various types of cultured cells in the absence of metabolic activation. Only three of these studies for chromosomal aberrations included an exogenous metabolic activation system. Of these, one, using Syrian hamster embryo cells, found an increase in aberration frequency. Weak effects were detected for the induction of aneuploidy and mitotic division aberrations in Chinese hamster lung cells. 

RLV/Fischer rat assay without the addition of an exogenous metabolic activation system. In a single study, mouse JB6 epidermal cells were transformed by di(2-ethyl-hexyl) phthalate without activation and in one of two studies a weak response was reported in the CSHIOT A cell transformation assay with di(2-ethylhexyl) phthalate in either the absence or presence of exogenous metabolic activation. BALB/c-3T3 cells were not transformed by di(2-ethylhexyl) phthalate with or without metabolic activation. Di(2-ethylhexyl) phthalate inhibited gap-junctional intercellular communication in Chinese hamster V79 cells in six of seven studies, but not in one study of liver cells of cynomolgus monkeys in vivo. Di(2-ethylhexyl) phthalate treatment of Syrian hamster embryo cells in a two-stage exposure with 12-O-tetradecanoylphorbol 13-acetate resulted in superinduction of ornithine decarboxylase, an early event in morphological transformation no effect was seen after a one-stage treatment with di(2-ethylhexyl) phthalate alone. 

A-Nitrosodiethanolamine was mutagenic to Salmonella typhimurium in most assays in the presence of exogenous metabolic activation systems and in some assays in the absence of such systems. 

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